The employment of retrovirus derived vectors in biotechnological applications has been standard practice for many years. For example, early retrovirus-derived vectors are described in Wei et al., J. Virol., 39:935-44 (1980) and Shimotohno et al., Cell, 26:67-77 (1981). Retroviruses are single-stranded RNA viruses. During an infection process in a host subject, such as a human, the RNA viruses are reverse transcribed into double stranded DNA. The double stranded DNA is subsequently integrated into the host cell DNA, and the virus becomes a permanent part of the host cell DNA. Once integrated, the virus is capable of expression of more viral RNA as well as the proteins that make up the virion. As retroviruses are usually not lytic, the retrovirus can continue to produce virus particles that bud from the surface of the cell.
Typically, modern retroviral vectoring systems consist of (1) RNA molecule(s) bearing cis-acting vector sequences needed for transcription, reverse-transcription, integration, translation and packaging of viral RNA into the viral particles, and (2) helper virus particles, budding from vector producer cells (VPCs), which express the trans-acting retroviral gene sequences (as proteins) needed for production of virus particles. By separating the cis- and trans-acting vector sequences completely, the virus is unable to maintain replication for more than one cycle of infection. The trans-acting vector sequences make empty virions (viral particles), whereas cis-acting vector sequences are capable of perpetuation or duration, only in the presence of the helper particles. Thus, cis-acting vector sequences and retroviral helper cells are the two essential components of modern retroviral vectoring systems.
Retrovirus-derived vectors (RVs) have been used in the majority of gene therapy clinical trials for a variety of reasons. For example, RVs can permanently integrate and express foreign genes, thus overcoming the problem of transient (short-term) expression, which is a significant problem of DNA transfection. Retroviruses, however, also suffer from several significant drawbacks. For example, retroviruses usually infect only dividing cells, and have a lower titer than some DNA viruses, such as adenovirus 5-derived vectors having titers of  greater than 1011 transducing units/milliliter (TU)/ml. In addition, genetic recombination or xe2x80x9ccross-overxe2x80x9d can occur during replication (or at the DNA level), which can lead to outbreaks of replication competent retrovirus (xe2x80x9cRCRxe2x80x9d). RCRs occur as a result of regenerating the complete viral genome by genetic recombination. There are at least two different mechanisms by which this can occur. First, similar or identical overlapping nucleic acid sequences present on two separate DNA molecules (i.e., vector and helper sequences) can genetically recombine. Second, two separate RNA strands can serve as templates for cDNA synthesis during replication of the vector/virus, and genetic recombination can occur during DNA synthesis, leading to RCR. An additional confounding factor are the endogenous retroviral gene sequences that are present in the genome of the cells in which the virus is replicating. These retroviral gene sequences provide an additional source for generating RCR. Thus, it is important to separate the cis- and trans-acting sequences completely (i.e., no sequence overlap), and to provide a host cell genome that is devoid of closely related endogenous viral genes.
Unfortunately for most genetic therapies, RCR outbreaks have been detected in  greater than 15% of all lots of manufactured vectors tested prior to clinical trials. RCR are potentially lethal to primates and humans, and are therefore prohibited by the regulatory authorities. Significantly, the cost for detecting RCR can reach up to $100,000 per clinical batch. Often, however, RCR outbreaks often occur late in a vector production run as the VPCs are expanded. This suggests that a last minute outbreak might not be detected unless all of the clinical supernatant was tested. Thus, there is an xe2x80x98uncertainty principlexe2x80x99 that prevents complete assurance of catching the outbreak.
Still more unfortunately, many genetic therapy patients are immunecompromised (such as AIDS patients), and have reduced host defenses against oncogenic viruses, should an outbreak of RNA tumor virus occur. Clearly, this result presents many potential dangers. For example, an onco-retrovirus vector and the HIV retrovirus could infect the same cell. This mixed infection would most likely result in hybrid retrovirus particles containing both HIV-tropic and murine leukemia virus (xe2x80x9cMLVxe2x80x9d)-tropic particles (ie., the hybrid particles can share their host ranges, enabling a broader scope of infection). Such mixed pseudotype infections have the expected, expanded cellullar tropism (Lusso et al., Science, 247:848-852 (1990)). MLV-tropisms include the so-called amphotropic (or 4070A strain) and gibbon ape leukemia virus (GALV)-tropic viruses, which are each capable of infecting a wide variety of human cells (as opposed to the primarily T-cell tropism of the native HIV-1 virus). Thus, a mixed virus infection could lead to HIV-1 infection of an expanded human cell repertoire, and possibly to a situation where the virus spreads like a common infection.
Of equal concern would be a genetic recombination event between MLV and HIV-1, wherein the HIV-1 envelope acquires a recombinant tropism, such as amphotropism or GALV-tropism. This could potentially provide a new HIV virus that could spread easily between humans, infecting most cells. It might be possible for only a small portion of the murine envelope glycoprotein gene to be present in order for this situation to occur (thus it might not disrupt other essential HIV-1 genes).
Therefore, a need exists for safe and efficient vectors for the transmission of genetic materials in a mammal. The genetic elements utilized in such vectors should be able to transmit genetic material that can be employed in gene therapy and cell therapy protocols as well as other biotechnological applications.
The present invention provides a chimeric viral packaging signal that can be employed in a vector for transmission of genetic material. The packaging signal contains an essential packaging nucleic acid sequence isolated from a mammalian type C retrovirus that is functionally joined to at least one non-essential packaging nucleic acid sequence that is isolated from a murine VL30 nucleic acid sequence. Additionally, the non-essential packaging nucleic acid sequence lacks a gag gene sequence.
Preferably, a murine leukemia virus is the mammalian type C retrovirus source for the essential packaging nucleic acid sequence. Additionally, the packaging signal can further contain at least one long terminal repeat nucleic acid sequence. The long terminal repeat nucleic acid sequence can be isolated from several possible sources, such as, a murine VL30 element, a retrovirus or a retrotransposon. Preferably, a packaging signal of the invention is employed in a retroviral vector.
The invention also provides a chimeric viral packaging signal that contains a long terminal repeat nucleic acid sequence isolated from a type C retrovirus or retrotransposon and operably linked to an essential packaging nucleic acid sequence. The essential packaging sequence is typically isolated from a mammalian type C leukemia virus and is operably linked to at least one non-essential packaging nucleic acid sequence. Preferably, the non-essential nucleic acid sequence is isolated from a murine VL30 nucleic acid sequence and lacks a gag gene sequence. Additionally, the prepared packaging signal is capable of packaging viral RNA or vector RNA into a retroviral capsid.
In one embodiment, the long terminal repeat nucleic acid sequence employed in a packaging signal of the invention is isolated from a murine retrovirus, a murine VL30 nucleic acid sequence, a retrotransposon, a simian retrovirus, an avian retrovirus, a feline retrovirus, a lentivirus, an avian retrovirus or a bovine retrovirus. The essential packaging nucleic acid sequence is isolated from a murine leukemia virus and contains at least a portion of SEQ ID NO: 22. Typically, the non-essential packaging nucleic acid sequence is isolated from a VL30 element that is obtained from either NVL-1, NVL-2, NVL-3, BVL-1, VL3, VM1, TLEV, VLSI, PB10, PA2, VL11, VLOV1 or VLOV2. Preferably, the non-essential packaging nucleic acid sequence contains at least a portion of SEQ ID NO: 23.
The invention also provides a packaging signal that contains an essential packaging nucleic acid sequence having a 52 basepair duplication between a minus strand primer binding site and a splice donor site. An exemplary vector containing this packaging signal is the vector, pVLMB1 (FIG. 5).
A retrovirus is a single stranded, diploid RNA virus that replicates by means of reverse transcriptase and a retroviral virion. A retrovirus can be replication-competent or non-replication competent.
A retrotransposon (RTN) is a cellular mobile genetic element with long terminal repeats.
A VL30 is a virus-like, 30S RNA (retrotransposon) expressed in the cells of various vertebrate species. It is typified by LTRs, primer binding sites, and encapsidation signals. Although it does not contain intact viral genes, it is a viral parasite, or xe2x80x98selfish DNAxe2x80x99 that uses retroviral infections to be transmitted from cell to cell or from organism to organism. VL30s are usually found as integrated, 5 kb DNA sequences found in multiple copies within the cell genome. The 5 kb genomic DNA sequence consisting of LTRs and a stuffer fragment that can be replaced by foreign genes. VL30 vectors use cis-elements from the VL30 genome, or a synthetic equivalent.
An LTR is a long terminal repeat. LTRs are sequences found flanking i.e., positioned 5xe2x80x2 and 3xe2x80x2, retroviruses and retrotransposons such as VL30. The LTR sequence is typically at least several hundred bases long, bearing inverted repeats at its termini (often starting with TGAA . . . and ending with TTCA), and flanked with short direct repeats duplicated within the cell DNA sequences flanking the insertion site. The short inverted repeats are involved in integrating the full length viral, retrotransposon, or vector DNA into the host genome. The integration sequence is sometimes called att, for attachment. Inside the LTRs reside three distinct subregions: U3 (the enhancer and promoter region, transcribed from the 5xe2x80x2-LTR), R (repeated at both ends of the RNA), and U5 (transcribed from the 5xe2x80x2-LTR). The LTR and its associated flanking sequences (primer binding sites, splice sites, dimerization linkage and encapsidation sequences) comprise the cis-acting sequences of the retro-element or vector. Sources of LTR nucleic acid sequences, i.e., nucleic acid fragments or segments, include, but are not limited to murine retroviruses, murine VL30 sequences, retrotransposons, simian retroviruses, avian retroviruses, feline retroviruses, lentiviruses. avian retroviruses and bovine retroviruses.
A virion is a virus particle. In the case of retrovirus particles, the virion consists of an envelope with its characteristic envelope glycoprotein (capable of attaching to the recipient [target] cells) and a proteinaceous capsid, enclosing the viral or vector RNA together with its associated nucleocapsid protein. Also at the core of the virion is the RNA dimer, together with two copies of tRNA (primer) hydrogen-bonded to the viral or vector RNA. In addition to these virion-associated nucleic acid molecules, additional molecules of reverse transcriptase (an RNA directed DNA polymerase) catalyze replication, RNAse H [hybrid] activity, and integration [into cell DNA] via an integrase activity associated with the carboxyl terminus of the protein.
Murine leukemia virus (MLV) is a simple onco-retrovirus (a mammalian type C retrovirus), having three structural genes: gag (core particle), pol (reverse transcriptase), and env (envelope glycoprotein). These genes reside within the 10 kb backbone provirus, flanked by LTRs, primer binding sites, and a packaging signal.
Mammalian Type C retroviruses are viruses that are capable of infecting mammals and are a subgroup of the family retro viridae.
Retroviral helper cells are cultured cells engineered so as to produce retroviral virions, but not to package or transmit the RNA xe2x80x9chelperxe2x80x9d sequences which encode the viral proteins. Thus, a retroviral or retrotransposon vector (or a chimeric vector) can be transmitted by the helper cells.
Vector producer cells (VPCs): helper cells that are expressing vector particles.
Packaging signals (encapsidation signals or xcexa8, for Packaging) contain both essential and non-essential nucleic acid sequences that are responsible for packaging (into virions) viral, retrotransposon, or vector derived RNA, and for transmission efficiency. Although this function is poorly understood, it is known to consist of multiple sites. One site is near to (or overlapping with) a canonical splice donor-like sequence near the (xe2x88x92)PBS. Another potentially important site is the dimerization linkage site (dl) which is responsible for the non-covalent joining of two copies of the RNA near the 5xe2x80x2-LTR. Yet another potentially important site is the packaging hairpin, or GACG loop. This sequence, together with several palindromic bases adjoining it, are highly conserved. Occasionally, an Aat2 restriction endonuclease site located asymmetrically within the loop is also conserved (as in the case of MLV and VL30). This serves as a site for artificially joining the MLV xcexa8 and the VL30 xcexa8+ sequences. Yet another important sequence is the extended 3xe2x80x2-region known as xcexa8+. In the case of MLV, this is the first approximate one-third of the retroviral gag gene sequence. In mouse VL30, it usually consists of some direct, overlapping repeats and other sequences 3xe2x80x2 in direction from the Aat2 site. At least in lentiviruses, LTR sequences may also be involved in packaging, and in onco-retroviruses (such as ASLV), some packaging sequences may be located elsewhere in the genome. It is considered axiomatic that all sequences included in a retrovirus, retrotransposon or vector may also affect packaging through other mechanisms, such as size, protein binding affinity or secondary structure.
A gag gene sequence is a linear piece of DNA encoding at least one viral structural protein. FIG. 4 shows a particular gag gene sequence ( basepairs 621 to basepairs 2237).
An xe2x80x9cessential packaging nucleic acid sequencexe2x80x9d, is a nucleic acid sequence isolated from a mammalian type C retrovirus. The essential packaging sequence contains a splice donor site, a dimerization sequence and a region of secondary structure, such as a stem loop structure. The stem loop structure typically contains the sequence GACG in the loop portion and is usually referred to as an essential packaging loop. In some instances, the highly conserved essential packaging loop, or xcexa8 loop, also contains an Aat2 restriction enzyme site, enabling the essential packaging loop to be joined or operably linked to a non-essential packaging sequence at the Aat2 restriction enzyme, which is a common site between these two sequences. A preferred essential packaging nucleic acid sequence is isolated from a murine leukemia virus (xe2x80x9cMLVxe2x80x9d), and more preferably from the Moloney strain MLV (xe2x80x9cMoMLVxe2x80x9d).
A xe2x80x9cnon-essential packaging nucleic acid sequencexe2x80x9d is composed of a nucleic acid sequence that extends beyond the 3xe2x80x2-terminus of the essential packaging loop that are not required for specific packaging into virus particles, but which enhance or add to the infectious titer of a virus or vector when present. Because these nucleic acid sequences are non-essential, the xcexa8+ sequences are more variable than xcexa8 sequences, and there is no distinct 3xe2x80x2-terminus sequence boundary. For example, in MLV, the xcexa8+ sequences are thought to extend from about basepairs 620 to about basepairs 1040 of the MLV genome (Bender et al., J. Virol., 61:1639-1646 (1987)). These xcexa8+ sequences include about a third of the gag gene sequence. In mouse VL30, the 3xe2x80x2-terminus of the xcexa8+ region has not been completely defined, but it may include regions extending from the GACG loop to the end of the VL30 genome. However, the region within 700 basepairs of the GACG loop is of particular interest as it appears to contain powerful xcexa8+ sequences as well as inhibitory sequences that can be removed to improve the titer of a packaging signal. The non-essential packaging nucleic acid sequence is typically an isolated VL30 nucleic acid sequence obtained from a rat or mouse. Preferably, an isolated mouse VL30 nucleic acid sequence is employed in a packaging signal of the invention.
Isolated, as used herein, means that a naturally occurring nucleic acid sequence, DNA fragment, DNA molecule, coding sequence, or oligonucleotide is removed from its natural environment, or is a synthetic molecule or cloned product. Preferably, the nucleic acid sequence, DNA fragment, DNA molecule, coding sequence, or oligonucleotide is purified, i.e., essentially free from any other nucleic acid sequence, DNA fragment, DNA molecule, coding sequence, or oligonucleotide and associated cellular products or other impurities.
Vector designations preceded by a lower case xe2x80x98pxe2x80x99 indicate plasmid DNA (transfection), whereas vectors designated by all upper case letters indicate the virus (transduced) form.